1. Field of the Invention
The present invention relates to the regulation of ephrin-Eph receptor signaling mechanisms. Particularly, the present invention relates to the regulation of ephrinB3-EphA4 signaling mechanisms by targeting α-chimerin (α-chimaerin) in the signaling mechanisms.
2. Background Art
Ephrins are cell surface-bound proteins and are ligands for Eph receptors. Ephrin/Eph binding induces bidirectional signaling (see Noren, N. K. et al., (2004) Cell Signal. 16, 655-666 and Palmer, A. et al, (2003) Genes Dev. 17, 1429-1450). Ephrin/Eph signaling, which functions in short-range cell-to-cell communication usually through repulsive effects, plays a central role in neuronal circuit formation (see Palmer, A. et al, (2003) Genes Dev. 17, 1429-1450, Flanagan, J. G. et al., (1998) Annu. Rev. Neurosci. 21, 309-345, and Pasquale, E. B. (2005), Nat. Rev. Mol. Cell Biol. 6, 462-475). Downstream signaling mechanisms have been studied mostly through cell culture experiments. Particularly, important players are the Rho-family GTPases (Rho-GTPases), such as RhoA, Rac, and Cdc42, which are the key regulators of actin dynamics (see Noren, N. K. et al., (2004) Cell Signal. 16, 655-666, Etienne-Manneville, S. et al., (2002) Nature 420, 629-635, Luo, L. (2000), Nat. Rev. Neurosci. 1, 173-180, and Wahl, S. et al, (2000) J. Cell Biol. 149, 263-270). Rho-GTPases are directly activated by Rho-guanine nucleotide-exchange factors (Rho-GEFs) and are inactivated by Rho-GTPase-activating proteins (Rho-GAPs). Reports in recent years suggest that ephrin/Eph regulates Rho-GTPases via Rho-GEFs (see Cowan, C. W. et al., (2005) Neuron 46, 205-217, Irie, F. et al., (2002) Nat. Neurosci. 5, 1117-1118, Murai, K. K. et al., (2005) Neuron 46, 161-163, Ogita, H. et al., (2003) Circ. Res. 93, 23-31, Penzes, P. et al., (2003) Neuron 37, 263-274, Shamah, S. M. et al., (2001) Cell 105, 233-244, and Tanaka, M. et al., (2004) EMBO J. 23, 1075-1088). Among the numerous Rho-GEFs allegedly involved in ephrin/Eph, ephexin1 has been most studied. EphA receptors regulate growth-cone dynamics via ephexin1 in axon guidance (see Shamah, S. M. et al., (2001) Cell 105, 233-244 and Sahin, M. et al., (2005) Neuron 46, 191-204). In in vitro experiments, activation of RhoA induces growth-cone retraction, while activation of Rac and Cdc42 induces its extension (see Etienne-Manneville, S. et al., (2002) Nature 420, 629-635 and Luo, L. (2000), Nat. Rev. Neurosci. 1, 173-180). The binding of Eph and ephrin leads to activation of the GEF activity of ephexin1 towards RhoA, thereby causing growth-cone collapse (see Shamah, S. M. et al., (2001) Cell 105, 233-244). However, as ephexin1-knockout (KO) mice are normal (see Sahin, M. et al., (2005) Neuron 46, 191-204), the function of ephexin1 in vivo remains unknown. It is also noteworthy that, compared with the considerable attention given to Rho-GEFs, the possible involvement of Rho-GAPs in actin dynamics regulated by ephrin/Eph has been neglected.
The roles of ephrin/Eph in vivo have been studied using mouse reverse genetics. Particularly, ephrinB3→EphA4 forward signaling is extremely well-characterized. EphrinB3-KO and EphA4-KO mice have many common phenotypes, including a rabbit-like gait and abnormality in two major motor circuits: the corticospinal tract and the central pattern generator (CPG). Similar phenotypes are also displayed by EphA4KD/KD and EphA4FF/FF mice having the kinase activity of EphA4, but not by mice having ephrinB3 lacking its cytoplasmic domain (see Dottori, M. et al., (1998) Proc. Natl. Acad. Sci. USA 95, 13248-13253, Kullander, K. et al., (2003). Science 299, 1889-1892, Kullander, K. et al., (2001). Genes Dev. 15, 877-888, Kullander, K. et al., (2001) Neuron 29, 73-84, and Yokoyama, N. et al., (2001) Neuron 29, 85-97). Thus, it is apparent that ephrinB3→EphA forward signaling, but not EphA4→ephrinB3 reverse signaling, is essential for the formation of these motor circuits. CST axons, which regulate voluntary movements, arise from the motor cortex, then cross the midline at the medulla, and descend the contralateral spinal cord (see Giamino, S. et al., (1999) Brain Res. Dev. Brain Res. 112, 189-204 and Liang, F. Y. et al., (1991) J. Comp. Neurol. 311, 356-366). In wild-type mice, CST axons rarely cross back the midline (i.e., re-cross the midline) in the spinal cord, because ephrinB3 is anchored at the midline and transmits repulsive signals via EphA4 expressed on CST axon surface. In ephrinB3- or EphA4-knockout mice, due to a lack of repulsive ephrinB3/EphA4 forward signaling, many CST axons fail to stop at the midline and re-cross it (see Kullander, K. et al., (2001). Genes Dev. 15, 877-888 and Yokoyama, N. et al., (2001) Neuron 29, 85-97). Spinal CPGs are thought to generate left-right alternate stepping of limbs during walking (see Grillner, S. et al., (1985) Annu. Rev. Neurosci. 8, 233-261). Locomotor-like rhythmic activity alternating between the left and right sides can be induced in isolated wild-type spinal cords (see Nishimaru, H. et al., (2006) J. Neurosci. 26, 5320-5328), whereas the rhythmic activity of the two sides is synchronous in ephrinB3- or EphA4-knockout mice (see Kullander, K. et al., (2003). Science 299, 1889-1892). Aberrantly frequent midline crossing of EphA4-expressing CPG interneurons is thought to be responsible for such abnormality in the knockout mice (see Kullander, K. et al., (2003). Science 299, 1889-1892 and Kiehn, O., et al., (2004) Neuron 41, 317-321).